We theorize that excessive degradation of the fetal membrane extracellular matrix (ECM) by specific matrix metalloproteinases (MMPs) results in preterm premature rupture of the membranes (PROM). Active, inhibitor free MMP2 and 9 (gelatinase A and B respectively) can degrade the amniochorion basement membrane Type IV collagen to initiate rupture. This study examines the levels of the gelatinases and their natural inhibitors (tissue inhibitor of matrix metalloproteinases-TIMPs) in the amniotic fluid during PROM, preterm labor (PTL) and at term. A total of 51 AF samples were collected from the following groups of patients. Group 1: Women with PTL and no ROM (n = 16) Group 2: Women with PROM (n = 16) irrespective of labor status Group 3: Women at term with intact membranes undergoing cesarean delivery irrespective of labor status (n = 19). ELISA was used to assay MMP2, MMP9, TIMP1 and TIMP2 levels in the amniotic fluid. The active, TIMP free levels of MMP2 were quantitated by zymography followed by computerized densitometry. Active MMP9 was measured using a bioassay that specifically detects MMP9 activity. Statistical analysis was performed by Tukey-Kramer multiple comparison method. PROM is associated with increased MMP2 levels (mean 2125 ng/ml;) when compared with term (mean 1455 ng/ml; p < 0.01) or PTL where a non significant increase was seen (mean 1862 ng/ml; p = ns). MMP9 levels were higher in PROM (mean 15.03 ng/ml) than at term (mean 1.14 ng/ml; p < 0.001) or PTL (mean 3.75 ng/ml; p < 0.01). TIMP1 levels were slightly increased during PROM (mean 3143 ng/ml) compared to term (mean 1892 ng/ml; p < 0.05) pr PTL where a non significant change was seen (mean 2406 ng/ml; p = ns). TIMP2 levels were decreased in PROM (mean 98 ng/ml) compared with term (mean 176 ng/ml; p < 0.05) and PTL (mean 236 ng/ml; p < 0.001). Active, TIMP free MMP2 levels were increased during PROM (mean 233 pg/ml) compared to those at term (mean 132 pg/ml; p < 0.05) or PTL (mean 132 pg/ml; p < 0.05). Active forms of MMP9 were seen only during PROM (mean 632 pg/ml). Active, TIMP free forms of MMP2 and 9 are increased in the amniotic fluid of women with PROM. These MMPs can degrade the amniochorion basement membranes and other ECM components resulting in PROM.